Cancer cell – Fibroblast crosstalk via HB-EGF, EGFR, and MAPK signaling promotes the expression of macrophage chemo-attractants in squamous cell carcinoma

Summary Interactions between cells in the tumor microenvironment (TME) shape cancer progression and patient prognosis. To gain insights into how the TME influences cancer outcomes, we derive gene expression signatures indicative of signaling between stromal fibroblasts and cancer cells, and demonstrate their prognostic significance in multiple and independent squamous cell carcinoma cohorts. By leveraging information within the signatures, we discover that the HB-EGF/EGFR/MAPK axis represents a hub of tumor-stroma crosstalk, promoting the expression of CSF2 and LIF and favoring the recruitment of macrophages. Together, these analyses demonstrate the utility of our approach for interrogating the extent and consequences of TME crosstalk.


Figure S1 :
Figure S1: Generation and validation of CoCu8 / CoCu30 gene signatures, related to Figure 1.A) Flow chart description of the manuscript is provided.B) Strategy used to obtain CoCu30 gene signature.C) Venn diagram of the genes upregulated in the different datasets (top) and a table to summarize the genes constantly upregulated in the datasets (bottom) for cancer cells (left) and CAFs (right).D) Table

Figure S3 :
Figure S3: Multivariate analysis of CoCu8 overall survival, related to Figure 1.Forest plot showingHazard Ratios, 95% confidence interval and p value calculated using multivariate Cox regression from patients with HNSCC (HPV positive and negative), LUSC and CESC from the TCGA cohort.Variables include: age (continuous, years), sex (male vs female, except for CESC as all patients were female), clinical stage (categorical) and the CoCu8 signature (continuous variable).

Figure S4 :
Figure S4: Multivariate analysis of CoCu30 overall survival, related to Figure 1.Forest plot showing Hazard Ratios, 95% confidence interval and p value calculated using multivariate Cox regression from patients with HNSCC (HPV positive and negative), LUSC and CESC from the TCGA cohort.Variables include: age (continuous, years), sex (male vs female, except for CESC as all patients were female), clinical stage (categorical) and the CoCu30 signature (continuous variable).

Figure S5 :
Figure S5: Fibroblast abundance correlates with CoCu8 / CoCu30 gene signature in different squamous cell carcinoma datasets, related to Figure 1.A) Box plot analysis of CoCu8 expression in HNSCC, CESC and LUSC TCGA separated by first and last quartile of fibroblast abundance via MethylCIBERSORT deconvolution strategy.Independent Student's t-test.(Left)p-value<2.2e-16,(middle) p-value-0.028,(right), p-value=9.8e-6.B) Box plot analysis of CoCu8 expression in HPV positive and negative TCGA separated by first and last quartile of fibroblast abundance via MethylCIBERSORT deconvolution strategy.Independent Student's t-test.(Left) p-value=4.5e-7,(right) p-value=0.00016.C) Box plot analysis of CoCu30 expression in HNSCC, CESC and LUSC TCGA separated by first and last quartile of fibroblast abundance via MethylCIBERSORT deconvolution strategy.Independent Student's t-test.(Left), p-value<2.2e-16,(middle) p-value=0.0037,(right) p-value=7.2e-5.D) Box plot analysis of CoCu30 expression in HPV positive and negative TCGA separated by first and last quartile of fibroblast abundance via MethylCIBERSORT deconvolution strategy.Independent Student's t-test.(Left) p-value=8.4e-7,(right)p-value=2.8e-9.E-J) Correlation plot of different fibroblast subpopulations derived from Galbo et al. 9 with CoCu8 gene signature in LUSC TRACERx (E), HPV positive HNSCC TCGA (F), UK_HPV positive HNSCC (G) patients and with CoCu30 gene signature in LUSC TRACERx (H), HPV positive HNSCC TCGA (I), UK_HPV positive HNSCC (J) patients.The number inside the square represents the R, Spearman correlation coefficient.The color legend is shown at the bottom.All correlations are significant p-value<0.05.

Figure S7 :
Figure S7: Enrichment of CoCu8, CoCu30 and RAS84 gene signatures in different co-culture conditions, related to Figures 2, 3. A) Gene set enrichment analysis (GSEA) plot of CoCu8 gene signature (top) and CoCu30 (bottom) in co-culture indirect vs direct condition in A431 / VCAF2b transcriptomic dataset.NES and FDR are specified below each plot.B) Gene set enrichment analysis (GSEA) plot of RAS84 gene signature 28 in mono-culture and co-culture.NES and FDR are specified below each plot.

Figure S8 :
Figure S8: HB-EGF/RAS/MAPK activity in cancer cells and CAFs mono-culture vs co-culture, related to Figures 3, 4, 5. A) Correlation plot of CoCu8 and RAS84 expression levels in HNSCC HPV positive UK_HPV positive cohort.R is Spearman correlation coefficient.P-value=2.2e-16.B) Correlation table of different fibroblast subpopulations derived from Galbo et al. 9 with RAS84 gene signature in the independent cohort UK_HPV positive of HNSCC HPV positive patients.The number inside the square represents the Spearman R, correlation coefficient.The color legend is shown on the top.All correlations are significant at p-value<0.001.n=97.C) Heatmap of expression of the HBEGF in all the tested transcriptomic datasets.Every box represents the fold change difference of each gene for the corresponding cancer cell line in mono-culture when compared to its corresponding CAF cell line in mono-culture.D) Immunofluorescence staining of E-Cadherin and Hoechst 33342 for SCC154 monoculture for the indicated treatments after 48h.E) qPCR analysis of ACTA2, FAP, FN1 and LRRC15 genes in OCAF1 mono-culture for the indicated treatments after 48h.mRNA expression is reported as mean ± standard deviation (SD) fold change difference over non-treated (NT) condition.Genes have been normalized over the average of GAPDH, ACTB and RPLP0 housekeeping genes.n = 3 independent experiments.Two tailed paired Student's t-test.p-value is indicated.F) qPCR analysis of HBEGF gene in SCC154 after 96h of treatment with the indicated conditions.mRNA expression is reported as

Figure S9 :
Figure S9: HB-EGF effect on cytokine production and monocyte/macrophage enrichment related to Figure 5. A) qPCR analysis of LIF and CSF2 genes in OCAF1 and SCC154 mono-cultures for the indicated treatments after 48h.mRNA expression is reported as mean ± standard deviation (SD) fold change difference over non-treated (NT) condition for each cell type with LIF, while for CSF2 fold change difference is reported over Hb-EGF 10ng/ml treated sample.Genes have been normalized over the average of GAPDH, ACTB and RPLP0 housekeeping genes.n ≥ 5 independent experiments.Two tailed paired Student's t-test.p-value * is p-value<0.05,** is p-value<0.01,**** is p-value<0.0001.B) Correlation plot of HBEGF with LIF (left) and HBEGF with CSF2 (right) expression levels in HNSCC HPV positive TCGA dataset.R is Spearman correlation coefficient.(Left) p-value=5.3e-4,(right) p-value=0.0004.C) (Right) Box plot analysis of immune cell absolute score via MethylCIBERSORT deconvolution strategy in HNSCC HPV positive separated by first and last quartile of fibroblast abundance.Independent Student's t-test.p-value=0.008.(Left) Box plot analysis of immune cell absolute score via MethylCIBERSORT deconvolution strategy in HNSCC HPV positive separated by RAS84_0 and RAS84_max.Independent Student's t-test.p-value=0.0112.D) (Right) Box plot analysis of immune cell absolute score via MethylCIBERSORT deconvolution strategy in HNSCC HPV positive separated by first and last quartile of HBEGF expression.Independent Student's t-test.p-value=0/0013.(Left) Box plot analysis of immune cell absolute score via MethylCIBERSORT deconvolution strategy in HNSCC HPV positive separated by HBEGF expression.Independent Student's t-test.p-value ns is non-significant.

Table with Normalized
Enrichment Score (NES) values of different combinations of cancer cells and CAFs breast cancer cell lines from Rajaram et al. 21.Negative values represent enrichment towards coculture condition.Color legend is shown.E)Table with NES values of CoCu8 and CoCu30 gene signatures for cancer cells and endothelial cells mono-culture vs co-culture available from Stine et al.